PURPOSE: Increasing evidence demonstrates that the four and a half LIM domain (FHL) gene and its protein products have different functions in the progression of various malignancies. However, the role of FHL protein 2 (FHL2) in cervical cancer (CC) has not been fully elucidated. In this study, we investigated the prognostic value of FHL2 expression in human CC tissues and the potential molecular mechanisms through which FHL2 modulates CC cell proliferation and apoptosis.
MATERIALS AND METHODS: We measured FHL2 expression in CC cell lines and tissues by quantitative real-time polymerase chain reaction and Western blot assays. The effects of FHL2 knockdown on cell proliferation and apoptosis in two CC cell lines were examined using RNA interference, cell counting kit-8, Western blot and flow cytometry assays. Furthermore, we assessed phosphorylated protein kinase B (p-AKT) and phosphorylated mammalian target of rapamycin (p-mTOR) expression in two CC cell lines to determine whether the AKT/mTOR pathway is involved in the effects of FHL2 silencing on cell proliferation and apoptosis. Nude mice tumorigenicity experiments were also performed to evaluate the effects of FHL2 on HeLa cell growth in vivo.
RESULTS: We found that FHL2 was significantly upregulated in CC cell lines and tissues. According to survival curves, high FHL2 expression levels in patients were correlated with poor prognosis. Moreover, by decreasing p-AKT and p-mTOR protein levels, silencing FHL2 significantly inhibited cell proliferation and induced apoptosis. FHL2 knockdown also induced apoptosis by increasing the Bax-to-Bcl2 ratio. By contrast, FHL2 overexpression significantly promoted cell proliferation. Finally, decreased tumour growth in an in vivo animal model also demonstrated the tumour-suppressing effects of FHL2 knockdown.
CONCLUSION: Our findings indicate that FHL2 is an important prognostic factor in CC and that it plays a crucial oncoprotein role by promoting cell proliferation and inhibiting apoptosis in CC, possibly by targeting the AKT/mTOR pathway.

Breast cancer development and progression are influenced by insulin-like growth factor receptor 1 (IGF1R) and insulin receptor (InsR) signaling, which drive cancer phenotypes such as cell growth, proliferation, and migration. IGF1R and InsR form IGF1R/InsR hybrid receptors (HybRs) consisting of one molecule of IGF1R and one molecule of InsR. The specific signaling and functions of HybR are largely unknown, as HybR is activated by both IGF1 and insulin, and no cellular system expresses HybR in the absence of holo-IGF1R or holo-InsR. Here we studied the role of HybR by constructing inducible chimeric receptors and compared HybR signaling with that of holo-IGF1R and holo-InsR. We cloned chemically inducible chimeric IGF1R and InsR constructs consisting of the extracellular domains of the p75 nerve growth factor receptor fused to the intracellular β subunit of IGF1R or InsR and a dimerization domain. Dimerization with the drugs AP20187 or AP21967 allowed specific and independent activation of holo-IGF1R, holo-InsR, or HybR, resulting in activation of the PI3K pathway. Holo-IGF1R and HybR both promoted cell proliferation and glucose uptake, whereas holo-InsR only promoted glucose uptake, and only holo-IGF1R showed anti-apoptotic effects. We also found that the three receptors differentially regulated gene expression: holo-IGF1R and HybR up-regulated EGR3; holo-InsR specifically down-regulated JUN and BCL2L1; holo-InsR down-regulated but HybR up-regulated HK2; and HybR specifically up-regulated FHL2, ITGA6, and PCK2. Our findings suggest that, when expressed and activated in mammary epithelial cells, HybR acts in a manner similar to IGF1R and support further investigation of the role of HybR in breast cancer.

Four-and-a-half LIM protein2 (FHL2) is a member of the LIM-only protein family, which plays a critical role in tumorigenesis. We previously reported that FHL2 is upregulated and plays an oncogenic role in glioblastoma (GBM), the most common and aggressive brain tumor. GBM is also marked by amplification of the epidermal growth factor receptor (EGFR) gene and its mutations, of which EGFRvIII is the most common and functionally significant. Here we report that FHL2 physically interacts with the wild-type EGFR and its mutated EGFRvIII form in GBM cells. Expression of FHL2 caused increased EGFR and EGFRvIII protein levels and this was due to an increase in protein stability rather than an increase in EGFR mRNA expression. In contrast, FHL2 knockdown using RNA interference reduced EGFR and EGFRvIII protein expression and the phosphorylation levels of EGFR and AKT. Consistent with these features, EGFR expression was significantly lower in mouse FHL2-null astrocytes, where reintroduction of FHL2 was able to restore EGFR levels. Using established GBM cell lines and patient-derived neurosphere lines, FHL2 silencing markedly induced cell apoptosis in EGFRvIII-positive cells. Targeting FHL2 significantly prevented EGFRvIII-positive GBM tumor growth in vivo. FHL2 expression also positively correlated with EGFR expression in GBM samples from patients. Taken together, our results demonstrate that FHL2 interacts with EGFR and EGFRvIII to increase their levels and this promotes glioma growth, representing a novel mechanism that may be therapeutically targetable.

Angiogenesis is associated with prostate cancer (PCa) development and progression. Aberrant expression of C-terminal binding protein (CtBP)2 has been observed in PCa, but whether its change in expression plays a significant role in angiogenesis has not been completely characterized. we attempted to integrate and analyze the genome-wide association study (GWAS) of follicle stimulating hormone receptor (FSHR) and CtBP2, the Cancer Genome Atlas (TCGA) data and CtBP2 binding data in CistromeMap (18) to explore the mechanism of CtBP2 in PCa, and performed pathway enrichment analysis. We revealed that the top 6 pathways were closely related with angiogenesis. We used siRNA and overexpression plasmids to silence and overexpress CtBP2 expression. Altered expression of CtBP2 affected the expression of VEGFA, FSHR, FHL2 and SMAD3 which are closely related with angiogenesis. In addition, silencing of CtBP2 markedly increased the apoptosis of PCa cells in vitro, and decreased the expression of IL-8, AT2R, CCND1 and MMP9 which are associated with cancer progression. These results highlight the association between CtBP2 and angiogenesis in PCa and indicate that CtBP2 may be a potential therapeutic target for PCa.

Hyperactivation of the phosphatydil-inositol-3' phosphate kinase (PI3K)/AKT pathway is observed in most NSCLCs, promoting proliferation, migration, invasion and resistance to therapy. AKT can be activated through several mechanisms that include loss of the negative regulator PTEN, activating mutations of the catalytic subunit of PI3K (PIK3CA) and/or mutations of AKT1 itself. However, number and identity of downstream targets of activated PI3K/AKT pathway are poorly defined. To identify the genes that are targets of constitutive PI3K/AKT signalling in lung cancer cells, we performed a comparative transcriptomic analysis of human lung epithelial cells (BEAS-2B) expressing active mutant AKT1 (AKT1-E17K), active mutant PIK3CA (PIK3CA-E545K) or that are silenced for PTEN. We found that, altogether, aberrant PI3K/AKT signalling in lung epithelial cells regulated the expression of 1,960/20,436 genes (9%), though only 30 differentially expressed genes (DEGs) (15 up-regulated, 12 down-regulated and 3 discordant) out of 20,436 that were common among BEAS-AKT1-E17K, BEAS-PIK3CA-E545K and BEAS-shPTEN cells (0.1%). Conversely, DEGs specific for mutant AKT1 were 133 (85 up-regulated; 48 down-regulated), DEGs specific for mutant PIK3CA were 502 (280 up-regulated; 222 down-regulated) and DEGs specific for PTEN loss were 1549 (799 up-regulated, 750 down-regulated). The results obtained from array analysis were confirmed by quantitative RT-PCR on selected up- and down-regulated genes (n = 10). Treatment of BEAS-C cells and the corresponding derivatives with pharmacological inhibitors of AKT (MK2206) or PI3K (LY294002) further validated the significance of our findings. Moreover, mRNA expression of selected DEGs (SGK1, IGFBP3, PEG10, GDF15, PTGES, S100P, respectively) correlated with the activation status of the PI3K/AKT pathway assessed by S473 phosphorylation in NSCLC cell lines (n = 6). Finally, we made use of Ingenuity Pathway Analysis (IPA) to investigate the relevant BioFunctions enriched by the costitutive activation of AKT1-, PI3K- or PTEN-dependent signalling in lung epithelial cells. Expectedly, the analysis of the DEGs common to all three alterations highlighted a group of BioFunctions that included Cell Proliferation of tumor cell lines (14 DEGs), Invasion of cells (10 DEGs) and Migration of tumour cell lines (10 DEGs), with a common core of 5 genes (ATF3, CDKN1A, GDF15, HBEGF and LCN2) that likely represent downstream effectors of the pro-oncogenic activities of PI3K/AKT signalling. Conversely, IPA analysis of exclusive DEGs led to the identification of different downstream effectors that are modulated by mutant AKT1 (TGFBR2, CTSZ, EMP1), mutant PIK3CA (CCND2, CDK2, IGFBP2, TRIB1) and PTEN loss (ASNS, FHL2). These findings not only shed light on the molecular mechanisms that are activated by aberrant signalling through the PI3K/AKT pathway in lung epithelial cells, but also contribute to the identification of previously unrecognised molecules whose regulation takes part in the development of lung cancer.

Changes in blood epigenetic age have been associated with several pathological conditions and have recently been described to anticipate cancer development. In this work, we analyze a publicly available leukocytes methylation dataset to evaluate the relation between DNA methylation age and the prospective development of specific types of cancer. We calculated DNA methylation age acceleration using five state-of-the-art estimators (three multi-site: Horvath, Hannum, Weidner; and two CpG specific: ELOV2 and FHL2) in a cohort including 845 subjects from the EPIC-Italy project and we compared 424 samples that remained cancer-free over the approximately ten years of follow-up with 235 and 166 subjects who developed breast and colorectal cancer, respectively. We show that the epigenetic age estimated from blood DNA methylation data is statistically significantly associated to future breast and male colorectal cancer development. These results are corroborated by survival analysis that shows significant association between age acceleration and cancer incidence suggesting that the chance of developing age-related diseases may be predicted by circulating epigenetic markers, with a dependence upon tumor type, sex and age estimator. These are encouraging results towards the non-invasive and perspective usage of epigenetic biomarkers.

FHL2 is a multifunctional scaffolding protein; its expression is associated with poor prognosis in colorectal cancer. ADAM-17 is a metalloprotease implicated in ectodomain shedding. FHL2 regulates ADAM-17 plasma membrane localisation, and FHL2 deficiency leads to decreased activity of ADAM-17 in mouse macrophages. Presence and relationship of the ADAM-17/FHL2 complex with colorectal cancer progression is unknown. We studied FHL2 and ADAM-17 expression in several colon cancer cell lines by immunocytochemistry and western blot. To highlight the interaction between both molecules, we used the Duolink

The four and a half LIM domains 2 (FHL2) has been shown to play important roles in the regulation of cell proliferation, survival, adhesion, motility and signal transduction in a cell type and tissue-dependent manner. However, the function of FHL2 in ovarian physiology and pathology is unclear. The aim of this study was to determine the role and functional mechanism of FHL2 in the progression of ovarian granulosa cell tumors (GCTs). Immunohistochemical analysis indicated that FHL2 was overexpressed in GCT tissues. Cellular localization of FHL2 in GCT cells was cell cycle dependent. Knockdown of FHL2 suppressed GCT cell growth, reduced cell viability and inhibited cell migration. Consistently, ectopic expression of FHL2 in GCT cells with very low endogenous FHL2 promoted cell growth, improved cell viability and enhance cell migration. Importantly, overexpression of FHL2 promoted GCT progression in vivo. Mechanistic studies indicated that FHL2 regulates AKT1 gene expression in vitro and in vivo. Knockdown of FHL2 or AKT1 in GCT cell lines induced very similar phenotypes. Ectopic expression of constitutively active AKT1 rescued FHL2 knockdown-induced arrest of GCT cell growth and reduction of GCT cell viability, suggesting that FHL2 regulates GCT cell growth and viability through controlling AKT1 expression. Finally, co-immunoprecipitation and chromatin immunoprecipitation analyses indicated that FHL2 functions as a co-activator of NFκB and AP-1 to regulate AKT1 gene transcription. In conclusion, results from the present study indicate that FHL2 exerts its oncogenic action in GCT cells via controlling AKT1 gene expression. FHL2 is a promising target for the development of novel drugs against ovarian granulosa cell tumor.

Regulated intramembrane proteolysis of epithelial cell adhesion molecule (EpCAM) results in shedding of the extracellular domain (EpEX) and release of the intra-cellular domain (EpICD) into the cytoplasm. Released EpICD associates with FHL2, β-catenin and Lef-1 to form a nuclear complex and triggers oncogenic signaling. This study was conducted to examine the nuclear expression of EpICD in hepatocellular carcinoma (HCC) and to assess the role of EpICD in HCC. EpICD immunoexpression was examined in 100 cases of HCC using tissue microarrays and correlated with clinicopathological parameters. We also examined the role of EpICD in HCC using EpICD cDNA transfected HCC cell line and EpCAM silenced HCC cell line by small interfering RNA (siRNA). Nuclear expression of EpICD was observed in 19 of 100 (19%) cases. Nuclear expression of EpICD significantly correlated with nuclear expression of β-catenin, and Ki-67 labeling index. In addition, nuclear expression of EpICD was associated with higher histologic grade and advanced T category. Forced overexpression of EpICD in the HCC cell significantly increased the cell proliferation, migration and invasion. The overexpression of EpICD also increased the expression levels of the active form of β-catenin and c-myc and cyclin D1. In contrast, downregulation of EpCAM by siRNA decreased the cell proliferation, migration, invasion and the expression of active form of β-catenin, c-myc and cyclin D1. Our present data suggest that EpICD plays important roles in HCC progression by modulating expression of target genes of EpCAM.

The expression of immediate early response 3 (IER3), a protein with a short half-life, is rapidly induced by various cellular stimuli. We recently reported that IER3 induces the apoptosis of cervical cancer cells and that its expression is downregulated in patients with cervical cancer. However, the molecular mechanism involved in the rapid degradation of IER3 remains unknown. Here, we demonstrate that MDM2 is an E3 ligase that interacts with IER3 and promotes its ubiquitination, followed by proteasomal degradation. Polyubiquitination of the conserved lysine 60 of IER3 is essential for its degradation. In addition, four and a half LIM domains protein 2 (FHL2) binds to both IER3 and MDM2, allowing for efficient MDM2-mediated IER3 degradation by facilitating an association between MDM2 and IER3. Moreover, IER3 induces cell cycle arrest in cervical cancer cells and its activity is further enhanced in cells in which FHL2 or MDM2 was silenced, thereby preventing IER3 degradation. The E6 and E7 oncoproteins of human papilloma virus 18 regulated IER3 expression. FHL2 expression was significantly higher in the squamous epithelium of cervical carcinoma tissues than in non-cancerous cervical tissues, whereas cervical carcinoma expression of IER3 was downregulated in this region. Thus, we determined the molecular mechanism responsible for IER3 degradation, involving a ternary complex of IER3, MDM2 and FHL2, which may contribute to cervical tumor growth. Furthermore, we demonstrated that FHL2 serves as a scaffold for E3 ligase and its substrate during the ubiquitination reaction, a function that has not been previously reported for this protein.

Platinum-based combination chemotherapy is the standard treatment for advanced non-small cell lung cancer (NSCLC). While cisplatin is effective, its use is not curative and resistance often emerges. As a consequence of microenvironmental heterogeneity, many tumour cells are exposed to sub-lethal doses of cisplatin. Further, genomic heterogeneity and unique tumor cell sub-populations with reduced sensitivities to cisplatin play a role in its effectiveness within a site of tumor growth. Being exposed to sub-lethal doses will induce changes in gene expression that contribute to the tumour cell's ability to survive and eventually contribute to the selective pressures leading to cisplatin resistance. Such changes in gene expression, therefore, may contribute to cytoprotective mechanisms. Here, we report on studies designed to uncover how tumour cells respond to sub-lethal doses of cisplatin. A microarray study revealed changes in gene expressions that occurred when A549 cells were exposed to a no-observed-effect level (NOEL) of cisplatin (e.g. the IC10). These data were integrated with results from a genome-wide siRNA screen looking for novel therapeutic targets that when inhibited transformed a NOEL of cisplatin into one that induced significant increases in lethality. Pathway analyses were performed to identify pathways that could be targeted to enhance cisplatin activity. We found that over 100 genes were differentially expressed when A549 cells were exposed to a NOEL of cisplatin. Pathways associated with apoptosis and DNA repair were activated. The siRNA screen revealed the importance of the hedgehog, cell cycle regulation, and insulin action pathways in A549 cell survival and response to cisplatin treatment. Results from both datasets suggest that RRM2B, CABYR, ALDH3A1, and FHL2 could be further explored as cisplatin-enhancing gene targets. Finally, pathways involved in repairing double-strand DNA breaks and INO80 chromatin remodeling were enriched in both datasets, warranting further research into combinations of cisplatin and therapeutics targeting these pathways.

Four and a half LIM domains 2 (FHL2) is a protein of 279 amino acids in length containing four full LIM-domains and a half LIM-domain at the amino terminus. FHL2 is one transcriptional cofactor that can interact with many different proteins, such as AP-1, BRCA1, IGFBP, and integrin, and involved in organ differentiation, development, cell apoptosis, and carcinogenesis. Recent studies showed that FHL2 could play different roles acting as co-activator or corepressor in different cancer types, depending on the cell types involved. However, no report about FHL2 function in tongue squamous cell carcinoma (TSCC) is available to date. This study aims to determine the FHL2 expression and its biological functions in TSCC via in vitro and in vivo studies. Results show that FHL2 expression was associated with the pathological differentiation of TSCC samples through immunohistochemistry. FHL2 overexpression could stimulate cell proliferation, invasiveness, and metastases investigated by MTT, flow cytometry, Transwell and cell scratch methods. FHL2 could also elevate tumor-related molecule nuclear transcription factor-B (NF-кB) and β-catenin expression levels both at transcriptional and translational levels through real-time PCR and Western blot analyses. The in vivo nude mice experiment showed that the tumorigenicity of FHL2 overexpression group was significantly increased compared with control groups. These results suggest that FHL2 overexpression could contribute to the growth, proliferation, invasiveness, and metastasis of human tongue squamous cell carcinoma; furthermore, its function in TSCC might be related with the upregulation of NF-кB and β-catenin expressions.

Four-and-a-half LIM-domain protein 2 (FHL2) is a multifunctional scaffolding protein regulating signalling cascades and gene transcription. It shuttles between focal adhesions and the nucleus where it signals through direct interaction with a number of proteins including β-catenin. The multiplicity of molecular pathways affected by FHL2 suggests an important role in several physiological and pathological events. The function of FHL2 in cancer is particularly intriguing, since it may act as an oncoprotein or as a tumour suppressor in a tissue-dependent fashion. In this review we present the current knowledge on the role of FHL2 in carcinogenesis, with emphasis on the digestive tract. We discuss the overexpression of FHL2 in colorectal, gastric and pancreatic cancer, the downregulation in hepatocellular carcinoma and the role of FHL2 in epithelial-mesenchymal transition. We briefly look at the potential role of FHL2 in the tumoural microenvironment and discuss how FHL2 expression and function might influence cancer treatment. Before implementation of FHL2 as a biomarker by pathologists, antibody validation should, however, be carried out.

Understanding how cells use complex transcriptional programs to alter their fate in response to specific stimuli is an important question in biology. For the MCF-7 human breast cancer cell line, we applied gene expression trajectory models to identify the genes involved in driving cell fate transitions. We modified trajectory models to account for the scenario where cells were exposed to different stimuli, in this case epidermal growth factor and heregulin, to arrive at different cell fates, i.e. proliferation and differentiation respectively. Using genome-wide CAGE time series data collected from the FANTOM5 consortium, we identified the sets of promoters that were involved in the transition of MCF-7 cells to their specific fates versus those with expression changes that were generic to both stimuli. Of the 1,552 promoters identified, 1,091 had stimulus-specific expression while 461 promoters had generic expression profiles over the time course surveyed. Many of these stimulus-specific promoters mapped to key regulators of the ERK (extracellular signal-regulated kinases) signaling pathway such as FHL2 (four and a half LIM domains 2). We observed that in general, generic promoters peaked in their expression early on in the time course, while stimulus-specific promoters tended to show activation of their expression at a later stage. The genes that mapped to stimulus-specific promoters were enriched for pathways that control focal adhesion, p53 signaling and MAPK signaling while generic promoters were enriched for cell death, transcription and the cell cycle. We identified 162 genes that were controlled by an alternative promoter during the time course where a subset of 37 genes had separate promoters that were classified as stimulus-specific and generic. The results of our study highlighted the degree of complexity involved in regulating a cell fate transition where multiple promoters mapping to the same gene can demonstrate quite divergent expression profiles.

Castration-resistant prostate cancer (CRPC) is the main challenge for prostate cancer treatment. Recent studies have indicated that extending the treatments to simultaneously targeting different pathways could provide better approaches. To better understand the regulatory functions of different pathways, a system-wide study of CRPC regulation is necessary. For this purpose, we constructed a comprehensive CRPC regulatory network by integrating multiple pathways such as the MEK/ERK and the PI3K/AKT pathways. We studied the feedback loops of this network and found that AKT was involved in all detected negative feedback loops. We translated the network into a predictive Boolean model and analyzed the stable states and the control effects of genes using novel methods. We found that the stable states naturally divide into two obvious groups characterizing PC3 and DU145 cells respectively. Stable state analysis further revealed that several critical genes, such as PTEN, AKT, RAF, and CDKN2A, had distinct expression behaviors in different clusters. Our model predicted the control effects of many genes. We used several public datasets as well as FHL2 overexpression to verify our finding. The results of this study can help in identifying potential therapeutic targets, especially simultaneous targets of multiple pathways, for CRPC.

Acquired resistance to lapatinib is a highly problematic clinical barrier that has to be overcome for a successful cancer treatment. Despite efforts to determine the mechanisms underlying acquired lapatinib resistance (ALR), no definitive genetic factors have been reported to be solely responsible for the acquired resistance in breast cancer. Therefore, we performed a cross-platform meta-analysis of three publically available microarray datasets related to breast cancer with ALR, using the R-based RankProd package. From the meta-analysis, we were able to identify a total of 990 differentially expressed genes (DEGs, 406 upregulated, 584 downregulated) that are potentially associated with ALR. Gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the DEGs showed that "response to organic substance" and "p53 signaling pathway" may be largely involved in ALR process. Of these, many of the top 50 upregulated and downregulated DEGs were found in oncogenesis of various tumors and cancers. For the top 50 DEGs, we constructed the gene coexpression and protein-protein interaction networks from a huge database of well-known molecular interactions. By integrative analysis of two systemic networks, we condensed the total number of DEGs to six common genes (LGALS1, PRSS23, PTRF, FHL2, TOB1, and SOCS2). Furthermore, these genes were confirmed in functional module eigens obtained from the weighted gene correlation network analysis of total DEGs in the microarray datasets ("GSE16179" and "GSE52707"). Our integrative meta-analysis could provide a comprehensive perspective into complex mechanisms underlying ALR in breast cancer and a theoretical support for further chemotherapeutic studies.

The transcription factor Krüppel-like factor (KLF)8 plays an important role in the formation of several human tumors, including colorectal cancer. We recently identified four-and-a-half LIM protein 2 (FHL2) as a critical inducer of the epithelial-to-mesenchymal transition (EMT) and invasion. However, the molecular mechanism by which KLF8 affects FHL2-mediated tumor proliferation, EMT and metastasis remains unknown. Here, we showed that KLF8 overexpression promoted EMT and metastatic phenotypes. KLF8 expression was stimulated by transforming growth factor (TGF)-β1. Moreover, KLF8 acted as a potential EMT inducer by stimulating vimentin expression and inducing a loss of E-cadherin in stable KLF8-transfected cells. KLF8 overexpression induced a strong increase in FHL2 expression, and a positive correlation between the expression patterns of KLF8 and FHL2 was observed in CRC cells. Promoter reporter and chromatin immunoprecipitation (ChIP) assays demonstrated that KLF8 directly bound to and activated the human FHL2 gene promoter. However, siRNA-mediated repression of FHL2 in KLF8-overexpressing cells reversed the EMT and the proliferative and metastatic phenotypes. In vivo, KLF8 promoted FHL2-mediated proliferation and metastasis via orthotopic implantation. Taken together, this work identified KLF8-induced FHL2 activation as a novel and critical signaling mechanism underlying human breast/colorectal cancer invasion and metastasis.

The Four-and-a-half LIM (FHL)-only protein is a subfamily of protein members under the LIM-only protein family. These proteins are identified by their characteristic four and a half cysteinerich LIM homeodomain. Five members have been categorized into the FHL subfamily, which are FHL1, FHL2, FHL3, FHL4 and activator of CREM in testis (ACT) in human. FHL2 is amongst the most examined members within the family. Fhl2, the gene that code for the protein, is transcriptionally regulated by diverse types of transcription factors, for example, p53, serum response factor (SRF), and specificity protein 1 (Sp1). The expression of FHL2 is found in different tissues and organs and has been reported as a critical participant influencing the wide types of cancer such as breast cancer, gastrointestinal (GI) cancers, liver cancer and prostate cancer. The expression profile of FHL2 appeared to have a significant functional role in the carcinogenesis of these cancers which are mediated by different types of transcription factor including both tumor suppressors and inducers. In this review, we will first describe the molecular network governing FHL2 expression, which focus on the transcription factors conveying FHL2-initiated responses. In the second part, FHL2-linked cancers and the underlying molecular machinery will be discussed. Factors other than transcriptional regulation which may involve the cancer progression such as mutations of fhl2 and posttranslational modifications of the protein will also be mentioned.

Pilomatricoma is a tumour derived from hair matrix cells, which shows progressive keratin expression. Tumorigenesis is frequently associated with activating mutations in β-catenin gene inducing nuclear expression of β-catenin protein. The present study analysed the role of transforming growth factor-β1 (TGF-β1) and four-and-a-half LIM domain protein 2 (FHL2) in pilomatricoma in synopsis with their expression patterns in human anagen hair. Human anagen hair showed TGF-β1 and nuclear FHL2 expression in the outer root sheath layer separated from nuclear β-catenin staining, which was observed in cells of matrix and inner root sheath layers. Correspondingly, 41 out of 50 pilomatricomas showed co-labelling of TGF-β1 and nuclear FHL2 in tumour cells, which mostly lacked nuclear β-catenin expression. Tumoural proliferation (ki67) was associated with nuclear β-catenin staining but not with expression of nuclear FHL2. In early pilomatricomas, TGF-β1 expression was observed in few peripheral tumour cells showing absent or faint nuclear FHL2 co-staining. TGF-β1 expression extended in growing tumours going along with strong nuclear FHL2 co-labelling as well as progressive keratin 14 and keratin 1 expression. In vitro, cultured human keratinocytes showed weak to marked autocrine TGF-β1 expression; in case of enhanced TGF-β1 expression associated with keratin 10 staining. TGF-β1-treatment of cultured human keratinocytes induced nuclear and cytoplasmatic FHL2 staining as well as keratin 14 staining. Accordingly, siRNA-mediated FHL2 knockdown of TGF-β1-stimulated keratinocytes reduced keratin 14 staining. In conclusion, tumoural TGF-β1 secretion seems to induce nuclear translocation of co-factor FHL2 mediating progressive keratin expression in pilomatricoma.

PURPOSE: A c-Src inhibitor blocks oestrogen (E2)-induced stress and converts E2 responses from inducing apoptosis to growth stimulation in E2-deprived breast cancer cells. A reprogrammed cell line, MCF-7:PF, results in a functional oestrogen receptor (ER). We addressed the question of whether the selective ER modulator 4-hydroxytamoxifen (4-OHT) could target ER to prevent E2-stimulated growth in MCF-7:PF cells.
METHODS: Expression of mRNA was measured through real-time RT-PCR. Global gene expression profile was analysed through microarray. Transcriptome profiles were screened by RNA-sequencing.
RESULTS: Unexpectedly, both 4-OHT and E2 stimulated cell growth in a concentration-dependent manner. Expression profiling showed a remarkable overlap in genes regulated in the same direction by E2 and 4-OHT. Pathway enrichment analysis of the 280 genes commonly deregulated in MCF-7:PF cells by 4-OHT and E2 revealed functions mainly related to membrane, cytoplasm and metabolic processes. Further analysis of 98 genes up-regulated by both 4-OHT and E2 uncovered a significant enrichment in genes associated with membrane remodelling, cytoskeleton reorganisation, cytoplasmic adapter proteins, cytoplasm organelle proteins and related processes. 4-OHT was more potent than E2 in up-regulating some membrane remodelling molecules, such as EHD2, FHL2, HOMER3 and RHOF. In contrast, 4-OHT acted as an antagonist to inhibit expression of the majority of enriched membrane-associated genes in wild-type MCF-7 cells.
CONCLUSIONS: Long-term selection pressure has changed the cell population responses to 4-OHT. Membrane-associated signalling is critical for 4-OHT-stimulated cell growth in MCF-7:PF cells. This study provides a rationale for the further investigation of target therapy for tamoxifen resistant patients.

FHL2, a member of the four and one half LIM domain protein family, is a critical transcriptional modulator. Here, we identify FHL2 as a critical regulator of hematopoietic stem cells (HSCs) that is essential for maintaining HSC self-renewal under regenerative stress. We find that Fhl2 loss has limited effects on hematopoiesis under homeostatic conditions. In contrast, Fhl2-null chimeric mice reconstituted with Fhl2-null bone marrow cells developed abnormal hematopoiesis with significantly reduced numbers of HSCs, hematopoietic progenitor cells (HPCs), red blood cells and platelets as well as hemoglobin levels. In addition, HSCs displayed a significantly reduced self-renewal capacity and were skewed toward myeloid lineage differentiation. We find that Fhl2 loss reduces both HSC quiescence and survival in response to regenerative stress, probably as a consequence of Fhl2-loss-mediated downregulation of cyclin-dependent kinase-inhibitors, including p21(Cip) and p27(Kip1). Interestingly, FHL2 is regulated under the control of a tissue-specific promoter in hematopoietic cells and it is downregulated by DNA hypermethylation in the leukemia cell line and primary leukemia cells. Furthermore, we find that downregulation of FHL2 frequently occurs in myelodysplastic syndrome and acute myeloid leukemia patients, raising a possibility that FHL2 downregulation has a role in the pathogenesis of myeloid malignancies.

Four and a Half LIM protein 2 (FHL2) is a LIM domain only protein that is able to form various protein complexes and regulate gene transcription. Recent findings showed that FHL2 is a potential tumor suppressor gene that was down-regulated in hepatocellular carcinoma (HCC). Moreover, FHL2 can bind to and activate the TP53 promoter in hepatic cells. In this study, the activity of the two promoters of FHL2, 1a and 1b, were determined in the human embryonic kidney cell line HEK293 and the activation of these two promoters by p53 was investigated. Our results showed that the 1b promoter has a higher activity than the 1a promoter in HEK 293 cells but the 1a promoter is more responsive to the activation by p53 when compared with the 1b promoter. The regulation of FHL2 by p53 was further confirmed in liver cells by the overexpression of p53 in Hep3B cells and the knockdown of p53 in HepG2 cells. Combining promoter activity results of truncated mutants and predictions by bioinformatics tools, a putative p53 binding site was found in the exon 1a of FHL2 from +213 to +232. The binding between the p53 protein and the putative p53 binding site was then validated by the ChIP assay. Furthermore, the expression of FHL2 and TP53 were down-regulated in majority of HCC tumour samples (n = 41) and significantly correlated (P = 0.026). Finally, we found that the somatic mutation 747 (G→T), a hot spot mutation of the TP53 gene, is potentially associated with a higher expression of FHL2 in HCC tumour samples. Taken together, this is the first in-depth study about the transcriptional regulation of FHL2 by p53.

After intramembranous proteolysis-mediated loss of the extracellular domain of the epithelial cell adhesion molecule (EpEx) and release of an intracellular domain (EpICD) into the cytoplasm, EpICD sequentially associates with FHL2 to form a nuclear complex with β-catenin and Lef-1. This association induces gene transcription involved in the activation of the oncogenic potential of epithelial cell adhesion molecule (EpCAM). We examined the localization and expression of EpEx, EpICD and β-catenin in surgical specimens of extrahepatic cholangiocarcinoma (ECC) from 79 patients and focused on the relationship between nuclear expression of EpICD and β-catenin. We also examined the role of EpICD by transfecting the EpICD cDNA in cholangiocarcinoma (CC) cell lines. There was a significant correlation between the nuclear expression of EpICD and β-catenin in ECC tissues. Frequent nuclear co-localization of EpICD and β-catenin was observed in cancer cells forming the invasive front. Nuclear expression of EpICD also significantly correlated with histologic grade of tumor. Overexpression of EpICD in the CC cells significantly increased the cell growth and proliferation. The overexpression of EpICD in the CC cells also increased the expression levels of the active form of β-catenin and EpCAM target genes, such as c-myc and cyclin D1. Furthermore, the overexpression of EpICD significantly enhanced the migration and invasiveness of CC cells. Conversely, the inhibition of EpCAM in EpCAM-overexpressing cells by siRNA significantly decreased cell proliferation, migration and invasion. These results indicate that the spatial localization of EpICD and its mutual interaction with β-catenin may be important in ECC progression and invasion.

BACKGROUND: Mutations of PRF1 gene have been identified in familial hemophagocytic lymphohistiocytosis type-2 (FHL-2) patients, and it has been reported as the commonest gene defect causing FHL. Patients with severe perforin deficiency usually present within first 1 year of life and with severe clinical manifestations.
OBSERVATION: We report 4 cases of severe perforin deficiency presenting with delayed onset and unusual clinical presentations viz., B-cell acute lymphoblastic leukemia, the Hodgkin lymphoma, tuberculosis, and the Still disease. Three of these 4 cases showed a common heterozygous missense mutation (p.Trp129Ser). Two of these patients expired because of uncontrolled hemophagocytic lymphohistiocytosis, one patient had 3 relapses while on therapy and one patient was in remission on maintenance therapy.
CONCLUSION: This study shows variety of clinical manifestations of perforin deficiency and although the onset of hemophagocytic lymphohistiocytosis is delayed in these patients, the outcome remains poor as in classical severe perforin deficiency patients.

FHL2 (Four and a half LIM-only protein 2) has been identified as an oncogene in colon cancer and suppression of FHL2 induces cell differentiation and tumorigenesis in colon cancer cell lines. The aim of this study was to develop a novel and effective approach to knockdown FHL2, which can serve as a promising target of colon cancer therapy. Recombinant adeno-associated virus (rAAV) was generated bearing with FHL2-shRNA and transfected into LoVo cells. Cell cycle and growth were assessed. The interaction between FHL2 and G0/G1 cell cycle and growth was evaluated by flow cytometry, western blot analysis and WST-1 assay. We showed that suppression of FHL2 by rAAV-shRNA induced G0/G1 cell cycle arrest and inhibited cell growth. Apoptosis-related proteins and their activity was investigated at the same time. rAAV-FHL2‑shRNA activated intrinsic and extrinsic apoptotic pathways and increased cell susceptibility to apoptotic stimuli by 5-FU. Moreover, a xenograft model was established to explore rAAV-FHL2-shRNA with 5-FU mediated tumorigenesis in vivo. A strong anti-tumorigenic effect of rAAV-FHL2-shRNA was shown in nude mice and this antitumor effect was enhanced when combined with 5-FU treatment. These findings implicate FHL2 as a cell cycle and growth modulator and thus inhibit apoptosis in colon cancer cells. rAAV-shRNA-FHL2 may serve as a novel and potent therapeutic or 5-FU co-therapeutic agent for colon cancer.

Four-and-a-half LIM-only protein 2 (FHL2) is an important mediator in many signaling pathways. In this study, we analyzed the functions of FHL2 in nuclear factor κB (NF-κB) signaling in the liver. We show that FHL2 enhanced tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) activity in transcriptional activation of NF-κB targets by stabilizing the protein. TRAF6 is a binding partner of FHL2 and an important component of the Toll-like receptor-NF-κB pathway. Knockdown of FHL2 in 293-hTLR4/MD2-CD14 cells impaired lipopolysaccharide (LPS)-induced NF-κB activity, which regulates expression of inflammatory cytokines. Indeed, FHL2(-/-) macrophages showed significantly reduced production of TNF and interleukin 6 (IL-6) following LPS stimulation. TNF and IL-6 are the key cytokines that prime liver regeneration after hepatic injury. Following partial hepatectomy, FHL2(-/-) mice exhibited diminished induction of TNF and IL-6 and delayed hepatocyte regeneration. In the liver, NF-κB signaling orchestrates inflammatory cross talk between hepatocytes and hepatic immune cells that promote chemical hepatocarcinogenesis. We found that deficiency of FHL2 reduced susceptibility to diethylnitrosamine-induced hepatocarcinogenesis, correlating with the activator function of FHL2 in NF-κB signaling. Our findings demonstrate FHL2 as a positive regulator of NF-κB activity in liver regeneration and carcinogenesis and highlight the importance of FHL2 in both hepatocytes and hepatic immune cells.

Androgen receptor (AR) action is critical for prostate cancer (CaP) progression, but is not inhibited fully by available androgen deprivation therapy (ADT). One of the limitations to current ADT is that it targets all androgen action in CaP, and other, cells irrespective of clinical relevance. The resulting off-target effects are responsible for ADT associated side effects that affect negatively a patient's quality of life. Isolation of the AR-dependent events that drive CaP progression may lead to novel forms of ADT that are at least as effective but more selective. Here, an approach is described that starts from insights in the basic mechanism(s) by which AR regulates target gene expression to identify novel drugable targets downstream of AR. Exploration of the molecular events that underlie androgen regulation of the AR-associated coregulator FHL2 led to the isolation of a novel indirect mechanism of androgen action that is mediated by the secondary transcription factor Serum Response Factor (SRF). Using a combination of oligoarray and in silico analyses, an SRF-dependent fraction of AR action was identified that is enriched in CaP tissues, is able to discriminate between benign and malignant prostate, and correlates with aggressive disease and biochemical failure. The RhoA signaling axis, a well known upstream stimulator of SRF action that harbors drugable targets, conveyed androgen-responsiveness to SRF, and was activated in CaP where it correlates with increased CaP aggressiveness and poor outcome after surgery.

BACKGROUND: The molecular mechanisms that are involved in the growth and invasiveness of osteosarcoma, an aggressive and invasive primary bone tumor, are not fully understood. The transcriptional co-factor FHL2 (four and a half LIM domains protein 2) acts as an oncoprotein or as a tumor suppressor depending on the tissue context. In this study, we investigated the role of FHL2 in tumorigenesis in osteosarcoma model.
METHODOLOGY/PRINCIPAL FINDINGS: Western blot analyses showed that FHL2 is expressed above normal in most human and murine osteosarcoma cells. Tissue microarray analysis revealed that FHL2 protein expression is high in human osteosarcoma and correlates with osteosarcoma aggressiveness. In murine osteosarcoma cells, FHL2 silencing using shRNA decreased canonical Wnt/β-catenin signaling and reduced the expression of Wnt responsive genes as well as of the key Wnt molecules Wnt5a and Wnt10b. This effect resulted in inhibition of osteosarcoma cell proliferation, invasion and migration in vitro. Using xenograft experiments, we showed that FHL2 silencing markedly reduced tumor growth and lung metastasis occurence in mice. The anti-oncogenic effect of FHL2 silencing in vivo was associated with reduced cell proliferation and decreased Wnt signaling in the tumors.
CONCLUSION/SIGNIFICANCE: Our findings demonstrate that FHL2 acts as an oncogene in osteosarcoma cells and contributes to tumorigenesis through Wnt signaling. More importantly, FHL2 depletion greatly reduces tumor cell growth and metastasis, which raises the potential therapeutic interest of targeting FHL2 to efficiently impact primary bone tumors.

Studies investigating the associations between Cytochrome P4502E1 (CYP2E1) polymorphisms and colorectal cancer (CRC) risk report conflicting results. We conducted a meta-analysis to assess the association between CYP2E1 gene Rsa I/Pst I, Dral T/A and 96-bp insertion polymorphisms and CRC susceptibility. Two investigators independently searched the Medline, Embase, CNKI, Wanfang, and Chinese Biomedicine Databases. Summary odds ratios (ORs) and 95 % confidence intervals (95 % CIs) for CYP2E1 polymorphisms and CRC were calculated in a fixed-effect model (the Mantel-Haenszel method) and a random-effects model (the DerSimonian and Laird method) when appropriate. Ultimately, 12, 5, and 4 studies were found to be eligible for meta-analyses of Rsa I/Pst I, Dral T/A, and 96-bp insertion polymorphisms, respectively. Our analysis suggested that the variant genotype of Rsa I/Pst I were associated with a significantly increased CRC risk (c2/c2 vs. c1/c1, OR = 1.36, 95 % CI = 1.04-1.77; recessive model, OR = 1.35, 95 % CI = 1.04-1.75). Moreover, similar results were observed between CYP2E1 96-bp insertion polymorphism and CRC risk (dominant model, OR = 1.25, 95 % CI = 1.07-1.45), while no association was observed between CYP2E1 Dral T/A polymorphism and CRC susceptibility in any genetic model. No publication bias was found in the present study. This meta-analysis shows that CYP2E1 Rsa I/Pst I and 96-bp insertion polymorphisms may be associated with CRC risk. The CYP2E1 Dral T/A polymorphism was not detected to be related to the risk for CRC.

Estrogen signaling and epigenetic modifications, in particular DNA methylation, are involved in regulation of gene expression in breast cancers. Here we investigated a potential regulatory cross-talk between these two pathways by identifying their common target genes and exploring underlying molecular mechanisms in human MCF-7 breast cancer cells. Gene expression profiling revealed that the expression of approximately 140 genes was influenced by both 17β-estradiol (E2) and a demethylating agent 5-aza-2'-deoxycytidine (DAC). Gene ontology (GO) analysis suggests that these genes are involved in intracellular signaling cascades, regulation of cell proliferation and apoptosis. Based on previously reported association with breast cancer, estrogen signaling and/or DNA methylation, CpG island prediction and GO analysis, we selected six genes (BTG3, FHL2, PMAIP1, BTG2, CDKN1A and TGFB2) for further analysis. Tamoxifen reverses the effect of E2 on the expression of all selected genes, suggesting that they are direct targets of estrogen receptor. Furthermore, DAC treatment reactivates the expression of all selected genes in a dose-dependent manner. Promoter CpG island methylation status analysis revealed that only the promoters of BTG3 and FHL2 genes are methylated, with DAC inducing demethylation, suggesting DNA methylation directs repression of these genes in MCF-7 cells. In a further analysis of the potential interplay between estrogen signaling and DNA methylation, E2 treatment showed no effect on the methylation status of these promoters. Additionally, we show that the ERα recruitment occurs at the FHL2 promoter in an E2- and DAC-independent fashion. In conclusion, we identified a set of genes regulated by both estrogen signaling and DNA methylation. However, our data does not support a direct molecular interplay of mediators of estrogen and epigenetic signaling at promoters of regulated genes.